Abstract:
【Objective】The purpose of the study to clone a novel myeloid differentiation factor 88(MyD88) gene
MyD88-3 from the Pacific oyster(
Crassostrea gigas), the expression pattern of
CgMyD88-3 gene was analyzed in different tissues of
C. gigas under
Vibrio alginolyticus challenge, so as to provide a theoretical basis for exploring the role of MyD88 gene in the immune regulation and the healthy cultivation of
C. gigas. 【Method】Coding region(CDS) of
CgMyD88-3 gene was cloned. The bioinformatics analysis was analyzed by ProtScale, SWISS-MODEL and InterPro. Realtime fluorescence quantitative PCR was used to analyze the expression changes of
CgMyD88-3 gene in different tissues of
C. gigas. 【Result】The CDS length of
CgMyD88-3 gene was 537 bp, encoding 178 amino acids residues. The theoretical molecular weight of
CgMyD88-3 protein was 20.74 kD and the isoelectric point was 6.10. The sub-cellular localizations of the
CgMyD88-3 protein were predicted in the cytoplasm.
CgMyD88-3 was predicted to have five casein kinase II phosphorylation sites(
17TEED
20,
29SNLE
32,
76TQNE
79,
118TAND
121,
123TKED
126) and three protein kinase C phosphorylation sites(
26TMK
28,
148TAK
150,
169SEK
171).
CgMyD88-3 amino acids possessed only TIR(Toll/interleukin-1 receptor) domain but lacked Death domain.
CgMyD88-3 amino acid sequence had the highest similarity with CvMyD88-like amino acid sequence of
Crassostrea virginica, which was 100%. The result of the phylogenetic tree based on MyD88 amino acid sequence similarity showed that
CgMyD88-3 firstly clustered with C. virginica CvMyD88-like, and then with CgMyD88-T1 and CgMyD88-T2 of
C. gigas. The real-time fluorescence quantitative RT-PCR detection revealed that
CgMyD88-3was expressed in all six tissues of healthy C. virginica. The highest relative expression was found in the mantle, followed by gills, and in other tissues, the order was haemocytes>gonads>digestive glands, with the lowest relative expression level in the adductor. After infection by
Vibrio alginolyticus, the relative expression of
CgMyD88-3 was up-regulated in the adductor, digestive glands, haemocytes and gills. The expression of
CgMyD88-3 reached peak at 72 h in adductor, digestive glands and haemocytes, respectively, and peaked at 6 h in gills, while the expression of
CgMyD88-3 at other times was lower than 0 h after infection in the mantle. 【Conclusion】The present results show that
CgMyD88-3 is expressed in all tissues of healthy C. virginica, the highest relative expression is found in the mantle. Stimulation by V. alginolyticus obviously induces the up-regulation of
CgMyD88-3 expression, indicating that the
CgMyD88-3 may play an important role in the defense of C. virginica against external microbial and pathogen infections.